The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Two to three hundred million infections by malarial parasites occur every year worldwide, with 1-2 million deaths per year worldwide (1-2). The fact that these deaths occur in individuals who are critically ill at the time of admission to the hospital, that over 80% of deaths occur within the first 24 hours of admission to the hospital, and that antibiotic resistant malarial parasites are spreading, mandate the development of a preventative vaccine for malaria that is more potent than existing vaccines.
Malaria Life Cycle: Infection and Intrahepatic Phase.
Infection of a human subject is initiated when the female Anopheles mosquito bites the human subject leading to the deposition in the blood stream of mosquito saliva which is contaminated with the infectious sporozoites. These sporozoite forms travel through the blood to the liver where they infect hepatocytes (3-4). The sporozoites replicate and mature in the hepatocytes over 8-10 days which results in the production of a large number of merozoite daughter cells which are released into the bloodstream. The malarial parasite is masked from and undetectable by the immune response while they are within the hepatocytes. The merozoite daughter cells are released from the hepatocyte by wrapping themselves in the membrane of the hepatocyte as they bud off into the blood stream. This process may mask the merozoite from detection by the immune response system.
Malaria Life Cycle: Intraerythrocytic Phase.
Once inside red cells, the merozoites increase in number and mature successively into the “ring stage”, the pigmented tropozoite stage and the schizont-adhesion stage prior to red cell lysis which releases more merozoites into the blood stream. These merozoites infect more red cells and the cycle is repeated over and over again (4). This “asexual” replication phase lasts from 36-72 hours and ends in lysis of the red cell. Each cycle of replication, maturation and release requires 36 hours in Plasmodium falciparum, 48 hours in P. ovale and P. vivax, and 72 hours in P. malariae. The timing of this part of its cycle is the basis of the characteristic periodicity of the fevers and symptoms which are characteristic of malaria infections. Malarial adhesion proteins appear on the surface of the infected red cell. During this intraerythrocytic asexual replication phase, some of the merozoites develop into male and female sexual gametocytes that are taken up by the mosquito feeding on the blood. The malarial parasites are masked from the immune response while they are within the red cells.
Malaria Life Cycle: Erythrocytic Sequestration Phase.
In the case of Plasmodium falciparum, cell adhesion proteins are produced by the malarial parasite during the intraerythrocytic phase. One of these proteins, designated PfEMP1 (Plasmodium falciparum erythrocyte membrane protein, causes the infected red cells to adhere to each other, to uninfected red cells, to platelets, and to the luminal membrane of endothelial cells which line the small vessels of the body's organs (4-10). STEVOR and RIFIN are two other plasmodium encoded proteins which contribute to adhesion of infected red cells (11-12). The formation of these microaggregates of red cells generated by the PfEMP1 protein and other malarial adhesion proteins which appear on the infected red cell membrane then adhere to the endothelial surface, obstruct the flow of blood in the visceral microvasculature, which then leads to the symptoms of malaria: anemia, chills, fever, coma and jaundice (4). The PfEMP1 also interacts with platelets generating platelet mediated formation of red cell aggregates (5). This “sequestration” of the infected red cells as aggregates in the microvasculature, contributes to the survival of these cells since they are not subject to uptake and destruction by the reticuloendothelial cells of the liver and spleen. In addition, these red cell aggregates are responsible for the development in 1-2% of infected individuals of life-threatening consequences: thrombosis, acidosis, cerebritis, renal and hepatic failure, meningitis, pulmonary edema, and splenic rupture with hemorrhage. The aggregates of infected red cells can bind to the syncytiotrophoblast supporting the embryo during pregnancy.
Malaria Life Cycle: Replication in the Female Mosquito Salivary Glands.
The gametocytes (male and female) fertilize or fuse once in the salivary glands of the mosquito thus forming the zygote (ookinetes) which results in the development of sporozoites which are re-introduced into the blood of the next human subject who is infected.
Malaria Life Cycle: Aggregation Regulating Severity of Disease.
As stated above, only 1-2% of the infected individuals develop life threatening consequences of the sequestration. Rosetting of red cells in in vitro assays appears to correlate clinically with severity of disease. The following proteins have been shown to bind the PfEMP1 protein in the infected erythrocytes: CD36, ICAM-1, P-selectin, thrombospondin, PECAM-1/CD31, and Duffy like domains of several cellular receptors.
History of Malarial Vaccine Development by Others.
Most vaccines have attempted to use the CSP or fragments thereof, alone or attached to immunomodulatory agents, to induce an adaptive immune response in an attempt to reduce the frequency of clinically detectable malaria (13). These immunomodulatory proteins have included: the hepatitis B core antigen protein, the TLR agonist rEA, and the SLAM receptor adaptor protein EAT-2. Unfortunately, these vaccines have had limited potency (13-14). To achieve protection against malaria by CSP vaccines (the goal of which is to decrease the number of sporozoites which reach the intrahepatic phase), high titers of high affinity antibodies from long-lasting durable memory B cells are required (15).
The PfEMP1 Protein as Target for Composition/Vaccine Development.
The PfEMP1 protein, due to its exposure on the surface of the red cell membrane, and its central importance to the avoidance of destruction by the RE system, and due to the central role it plays in generating the life-threatening consequences of the disease, potentially could be an attractive target for composition/vaccine development. The difficulty in using the PfEMP1 as a target comes from the ability of the malarial parasite to switch among 60 variants of this protein, which are encoded by the “var” genes (16-17). The pathogenicity of a malarial organism can be correlated with specific forms of this gene (16-17). The development of adhesion reversing antibodies for passive administration at the time of admission of critically ill patients with malarial infections is a potentially important therapeutic approach which is under development. But treating established infections may not be as successful as prevention of the infection in the first place with vaccines.
Testing of the RTS,S Vaccine in 10 Normal Volunteers.
This vaccine, which was originally reported by Nussenzweigh (18-19), is based on the incorporation of CSP into RTS,S recombinant hybrid particles. These particles consist of a spontaneous assembly of RTS and S particles. The RTS is a fusion protein of 19 NANP CSP repeats and the hepatitis B surface antigen (HBsAg). This is co-expressed in yeast with free HBsAg, which is the S protein (20-24). This consists of amino acids 207-395 of the P. falciparum ND54/3D7 that is fused to the HBsAg protein. This approach has been shown to require adjuvants.
This vaccine induced a high level of protection against sporozoite challenge (20) in animal models. Ten normal human volunteers were vaccinated (5 with history of previous vaccinations to the hepatitis B virus and 5 without). Levels of circulating CSP specific CD8 effector T cells were thought to be important in suppressing the intrahepatic phase of the disease. It is possible that a significant portion of the immune response was against the hepatitis B surface antigen. CTLs specific for CSP peptides were not detected in these vaccines (20-25). The vaccine induced increases in the levels of RTS,S-specific CD4 T cells that released IFN-gamma within 12 hours of contract with the RTS,S-antigen. Lymphoproliferative responses were induced at low levels in a majority of test subjects. Antibody titers to the CSP NANP antigen were low even after 6 months, whereas the antibody titers to the hepatitis B surface antigen were 10 to 1000 fold higher.
Results of a Global Clinical Trial of the RTS,S Vaccine in Subjects Up to 17 Months of Age (26).
The RTS,S vaccine was given to children up to 17 months old. This generated protection in only 55% protection against acquisition of a detectable infection with malaria and 47% protection against acquisition of severe malaria. The fact that the vaccine only protected 55% of the vaccinated subjects may also reflect the low potency of the vaccine technology used (connecting the target antigen to the Hepatitis B surface core antigen).
Results of Clinical Trial of the RTS,S Vaccine in 6537 African Infants Between the Age of 6-12 Weeks Published On Line in the NEJM on Nov. 9, 2012 (27). The vaccine or a placebo was administered monthly for three administrations to 6537 African infants between the ages of 6-12 weeks along with other vaccinations. The primary endpoint of the trial was the proportion of subjects who experienced their first malarial illness during the 12 months following the third vaccination. The incidence of first malarial illnesses in the vaccine arm was 0.31/person-years and in the placebo group was 0.40/person-year for a vaccine efficacy of 30.1% (95% CI: 23.6, 36.1).
The efficacy with respect to the incidence of severe episodes of malaria was 26% in the treatment group. This result was described by the authors as a “modest” result. Despite this incomplete protection, 99.7% of the individuals who received three vaccinations, showed anti-CSP antibodies in their blood stream within 30 days of the last vaccination. The level of the antibody was 209 EU/ml. This suggests that the level of antibodies that were neutralizing in terms of infection of hepatic cells were too low for complete protection. This failed vaccine trial may also have arisen not only from the young age of the test subjects but also from the low potency of the vaccine.
Many factors can reduce the response of an individual to a viral infection or to the induction of an immune response to vaccination: chronic disease, chronic infection, cancer and advanced chronological age. Additional problems include: weak immunogenicity of the target antigen, qualitative or quantitative defects of CD4 helper T cells, defective response in the older aged population due to diminished expression of CD40L in activated CD4 helper T cells, or low levels of presentation of target antigens on Class I or II MHC in dendritic cells (DCs). Among individuals above the age of 55, less than 20% of individuals vaccinated with the yearly multi-valent particle inactivated influenza vaccine develop a fully positive immune response (28-31).
One explanation for this reduced response is the decrease in function of the immune system with age. For example, there is a decrease in the number of naïve, antigen unexposed CD4 and CD8 T cells. Additionally, the ratio of the naïve to memory CD8/CD4 cells decreases as the chronological age increases. Further, CD4 cells become impaired, acquiring both quantitative and functional defects, such as diminished levels of the CD40 ligand (CD40L) on the surface of CD4 cells as well as a temporal retardation of the rate at which CD40 ligand (CD40L) is expressed on the surface of the CD4 cells following activation. (32-33). Accordingly, the amount of antibody that an elderly system is able to generate will be lower following infection or conventional vaccination. The CD40L is important for the expansion of antigen specific CD8 effector T cells and antigen specific B cells in response to vaccination.
Development by Applicant of the TAA/ecdCD40L Vaccine Platform Which Can Overcome the Low Potency of Current Vaccine Strategies for Malaria. The vaccine strategy is based on the linkage of the target associated antigen (TAA) to the ecdCD40L. This fusion protein can be administered either as a protein, or as an expression vector carrying a transcription unit encoding the TAA/ecdCD40L (such as the Ad-sig-TAA/ecdCD40L adenoviral vector, or other viral vectors). The vaccine can also be administered as a vector prime followed in 7 and 21 days with sc injections of the TAA/ecdCD40L protein composition/vaccine. Alternatively, the vaccine can be administered as a TAA/ecdCD40L transcription unit inserted into a plasmid DNA expression vector. This vaccine was developed by the Applicant's laboratory to overcome the problems which can limit the potency and the degree of the immune response to vaccination (34-44).
Problems that Lead to Poor Response to Vaccination.
These problems include: weak immunogenicity of the target antigen, qualitative or quantitative defects of CD4 helper T cells, defective response in the older aged population due to diminished expression of CD40L in activated CD4 helper T cells, or low levels of presentation of target antigens on Class I or II MHC in dendritic cells (DCs). The CD40L is important for the expansion of antigen specific CD8 effector T cells and antigen specific B cells in response to vaccination.
TAA/ecdCD40L Composition/Vaccine Strategy of Applicant.
In order to circumvent such functional defects in the immune response, as well as increase the immunogenicity of the target associated antigens, Applicant's laboratory (34-44) designed the TAA/ecdCD40L vaccine strategy. There are four versions of this composition/vaccine: 1) One in which the TAA/ecdCD40L transcription unit is embedded in a replication incompetent adenoviral vector (Ad-sig-TAA/ecdCD40L) which is injected SC at 7 day intervals, 2) One in which the vector is used as an initial priming injection, followed by two sc injections of the TAA/ecdCD40L protein, 3) One in which the vaccine consists solely of the TAA/ecdCD40L protein which is injected 3 times at 7 day intervals, and 4) One in which the TAA/ecdCD40L is inserted into a plasmid DNA expression vector. The TAA is connected through a linker to the aminoterminal end of the ecd of the potent immunostimulatory signal CD40L.
Steps Involved in the Induction of an Immune Response by the TAA/ecdCD40L Composition/Vaccine Platform.
The attachment of fragments of the CSP (and any other TAA) to the CD40L accomplishes two things: 1) the binding of the TAA/ecdCD40L protein to the CD40 receptor on the DCs as well as on the B cells and T cells, activate these cells thereby promoting a potent immune response (34, 36, 38); 2) once the TAA/ecdCD40L protein is engaged on the CD40 receptor of the DC, the entire TAA/ecdCD40L protein is internalized into the DC in a way that allows Class I as well as Class II MHC presentation (34).
The activated TAA loaded DCs then migrate to the regional lymph nodes (41, 43) where they can activate and induce expansion of the TAA specific CD8+ effector T cells. These antigen specific CD8+ effector cells become increased in number in the lymph nodes (34, 36), egress from the lymph nodes into the peripheral blood. The antigen specific CD8 effector T cells exit the intravascular compartment and enter into the extra-vascular the sites of inflammation or infection (38). In addition to showing that this vaccine increases the antigen specific CD8+ effector T cells in the sites of inflammation (38), we have shown that the activation and expansion of the B cells by the TAA/ecdCD40L protein increases the levels of the TAA specific antibodies in the serum (38, 41-42).